The migration of young neurons from sites where they are generated into the positions where they establish the circuitry of the adult brain is a critical step in development. Defects in migration cause a host of human birth defects, ranging from severe mental retardation to subtle learning disabilities, as well as a large number of the epilepsies. Our lab has focused on understanding the genes that control migration, in the hope that insights into this key step in normal development. We use the migration of the cerebellar granule neuron as a model system to examine the molecular control of glial-guided neuronal migration. The establishment of neuronal polarity is a key step in initiating neuronal migration along the glial guide. Screens for genes that function in granule neuron migration revealed high levels of expression of the polarity signaling complex mPar6a neurons exiting the cycle and establishing polarity. In C. elegans, a set of 6 PAR proteins establish anterior/posterior asymmetries and control subsequent asymmetric cell divisions. PAR proteins are conserved throughout evolution. In Preliminary Studies (Solecki et al, 2004), we discovered that the mPar6a signaling complex is localized in the centrosome of migrating cerebellar granule neurons, where it coordinates the movement of the centrosome and the nucleus as the neuron migrates along the glial fiber. In the proposed research, we will study the other components of the mPar6a complex, aPKCzeta and Par3, in the polarity of migrating granule neurons. The role of mPar6a in cell division suggests that targeted loss of function mutants and shRNA experiments will not be feasible. We will therefore use a novel method developed by Roger Tsien to incorporate a genetic tag (TC) which binds the dye ReAshS, into mParGq and use chromophore-assisted light inactivation with a monochromatic laser to inactivate mParGa in the centrosome. For those experiments, we will generate TC-mPar6a BAC transgenic mice, enabling studies on granule cells and cortical neurons. In a final group of experiments, we will study a receptor/ligand system expressed in granule cells which interacts with the mParGa complex, the EphB ligands ephrin-B1 and ephrin-B2. Together, these experiments will provide novel information on the regulation of neuronal migration in cortical regions of developing brain. [unreadable] [unreadable]